Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.10.1 (ERK)
95,504 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Fragments of African green monkey (Cercopithecus aethiops) DNA (3.5 to 18.0 kilobases) were inserted downstream from the thymidine kinase (TK, tk) coding region in pTK206/SV010, a gene construct which lacks both copies of the hexanucleotide 5'-AATAAA-3' and contains a simian virus 40 origin of replication, allowing it to replicate in Cos-1 cells. No polyadenylated tk mRNA was detected in Cos-1 cells transfected by pTK206/SV010. The ability of simian DNA fragments to restore tk gene expression was examined by measuring the incorporation of [125I]iododeoxycytidine into DNA in Cos-1 cells transfected by pTK206/SV010 insertion derivatives. tk gene expression was restored by the insertion in 56 of the 67 plasmids analyzed, and the level of expression equaled or exceeded that obtained with the wild-type tk gene in 30 of these. In all plasmids examined that showed restoration of tk gene expression, polyadenylated tk mRNA of discrete size was detected. The sizes of these tk mRNAs were consistent with the existence of processing and polyadenylation signals within the inserted DNA fragments. The frequency with which inserted fragments restored tk gene expression suggests that the minimal signal for processing and polyadenylation is a hexanucleotide (AAUAAA or a similar sequence). LTK- cells were biochemically transformed to TK+ with representative insertion constructs. pTK206/SV010 transformed LTK- cells at a very low frequency; the frequency of transformation with insertion derivatives was 40 to 12,000 times higher.
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PMID:Preparation of a "functional library" of African green monkey DNA fragments which substitute for the processing/polyadenylation signal in the herpes simplex virus type 1 thymidine kinase gene. 630

After transfection of mouse mammary tumor virus (MMTV) proviral DNA into cultured cells, the DNA is transcribed in a glucocorticoid-sensitive fashion. The large terminal repeat (LTR) region of MMTV is 1,328 nucleotides long and contains the regulatory information necessary for the hormonal response. We have constructed a MMTV LTR-thymidine kinase (tk) chimeric gene and have tested the biological activity of molecules containing various deletions in the LTR after transformation of LTK- APRT- mouse cells. In the TK+ transformants, both a LTR- tk chimeric RNA and an authentic tk RNA are correctly initiated and transcribed. The synthesis of the chimeric RNA as well as that of the tk RNA is hormonally regulated. A plasmid containing 202 nucleotides of LTR DNA 5' to the RNA initiation site is fully sensitive to glucocorticoids; 50 nucleotides still cause a residual inducibility.
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PMID:Subfragments of the large terminal repeat cause glucocorticoid-responsive expression of mouse mammary tumor virus and of an adjacent gene. 630 28

Mouse LTK- cells (H-2k) were transfected with a series of recombinant plasmids consisting of the herpes simplex virus type 1 thymidine kinase (TK) gene linked to fragments of SV40 DNA coding for portions of SV40 T antigen in pBR322, and TK+ transformants (LTK+) were selected in HAT medium. The TK+ transformants were analyzed for SV40 transplantation rejection antigen (TrAg) at the cell surface by reacting them with cytotoxic lymphocytes (CTL) generated to SV40 TrAg in C3H/HeJ (H-2k) mice. The results indicated that the cells transformed by pVBETK-1 and synthesizing full size SV40 large T antigen were efficiently lysed by SV40 CTL. In addition, cells transformed by the plasmid pVBt1TK-1 and synthesizing a truncated 33 K T antigen were also found to be susceptible to lysis by the CTL. However, LTK+ cells that were transformed with the plasmid pVBt2TK-1 and which synthesized a truncated T antigen of 12.3 K did not provide a target for SV40 CTL nor did pVBETK-1-transformed cells that did not express any of the SV40 tumor antigens. Only the pVBETK-1-transformed cells that express 94 K T antigen were able to immunize mice against a challenge of syngeneic SV40-transformed cells. These results suggest that the TrAg expression at the cell membranes of transformed cells may be associated with the proximal half of SV40 T antigen.
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PMID:Biology of simian virus 40 (SV40) transplantation antigen (TrAg). IX. Analysis of TrAg in mouse cells synthesizing truncated SV40 large T antigen. 631 Aug 60

A series of cell lines was constructed by transformation of murine LTK- cells with a family of deletion mutants of the herpes simplex virus (HSV) thymidine kinase (tk) gene. These mutants, differing in the extent of 5' sequence flanking the coding region for tk, varied in the frequency with which they were able to convert tk- cells to the tk+ phenotype. Converted cell lines were analysed for tk DNA sequences, tk mRNA sequences, the 5' terminus of tk-specific transcripts and for their ability to respond to a signal provided in trans by infecting tk- virus (transactivation). The results of these analyses reveal that transformation efficiency correlates inversely with the extent of 5' flanking information. Thus mutants retaining less than 109 bp of 5' sequences transform less efficiently than those that retain at least 109 bp. Cell lines established by transformation with mutants retaining the proximal 109 bp contain relatively few copies of tk DNA whereas those which arose as a result of transformation with mutant DNA containing less than 109 bp generally contained multiple copies of tk DNA. Analyses of tk-specific transcripts revealed that cell lines derived from plasmids that transformed efficiently synthesized an mRNA which was indistinguishable by its size or 5' end from infected cell mRNA. Cell lines established by plasmids that were inefficient at transformation accumulated truncated mRNAs that initiated at aberrant start sites. The presence of the 5' 109 bp block was required for transformants to increase the level of tk mRNA and enzyme when infected with a tk- deletion mutant of HSV. We also show that transactivation does not alter the initiation site of the tk mRNA synthesized by transformants.
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PMID:Control of expression of the herpes simplex virus thymidine kinase gene in biochemically transformed cells. 631 67

In a previous study the BamHI-K fragment of Epstein-Barr virus DNA was shown to induce a nuclear antigen, Epstein-Barr virus nuclear antigen (EBNA), when cotransfected with the herpes simplex virus thymidine kinase gene into mouse LTK- cells. We have now inserted the BamHI-K fragment and a BamHI/HindIII subfragment, I1f , into shuttle vectors containing the origin of replication of simian virus 40. These plasmids have been introduced into COS-1, which are monkey kidney cells transformed by an origin-defective simian virus 40 genome. This expression system permitted rapid characterization of antigens, mRNAs, and proteins related to EBNA. The same-sized EBNA protein (approximately 78,000) was made after transfection with BamHI-K (5.2 kilobase pairs [kbp]) or the I1f subfragment (2.9 kbp). A deletion of about 600 bp occurred when the I1f fragment was propagated on the pSV2 plasmid in Escherichia coli. The deleted fragment gave rise to a smaller protein (approximately 52,000). These data provide evidence that EBNA is encoded by the 2.9-kbp I1f and is not an induced cellular protein. Nuclear antigen and polypeptide expression occurred equally well when the Epstein-Barr virus DNA was cloned on PSV2 -gpt or pSVOd . The latter plasmid lacks sequences allowing for efficient early gene transcription as well as splicing and polyadenylation signals which are present in pSV2 . Preliminary mapping of the EBNA gene transcripts demonstrated that two mRNAs (2.9 and 2.4 kilobases [kb]) are homologous to the I1f fragment. Taken together, the data suggest that the 2.9-kbp I1f fragment contains the structural gene for EBNA synthesis. COS-1 cells will thus provide a valuable system in which to analyze functional domains of the EBNA gene.
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PMID:Expression in COS-1 cells of Epstein-Barr virus nuclear antigen from a complete gene and a deleted gene. 632 12

A recombinant plasmid containing the thymidine kinase (TK) gene (pAGO; 6.36 kilobases) was reacted in vitro with (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene, an ultimate carcinogenic metabolite of benzo(a)pyrene. The covalent binding of the metabolite to the circular forms of pAGO was visible by a drastic change in their mobility during agarose gel electrophoresis. The 4% modified DNA was only partially restricted by different endonucleases. Modification and limited restriction were correlated to the biological activity by transfer of the plasmid (TK gene), modified and unmodified, to TK-deficient cells. Upon transfection of mouse LTK- cells with modified plasmid or modified TK gene, no or only a few TK-positive cells were obtained, in contrast to the formation of many colonies after transfection with the unmodified plasmid (gene). Benzo(a)-pyrene itself and phenanthrene oxide, a weakly reactive but noncarcinogenic chemical, did not induce this effect. The reactive diol-epoxides of noncarcinogenic benzo(a)acridine and carcinogenic benzo(c)acridine showed a weaker but similar decreasing effect on the formation of TK+ clones. This inhibition of transformation efficiency suggests inactivation of the gene by chemical modification. Our experimental approach challenges the repair capacity of the eukaryotic cell and thus renders the strategy suitable not only as a eukaryotic test for carcinogens but also as a tool for the study of carcinogenesis as aberrant gene expression.
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PMID:Inactivation of the thymidine kinase gene after in vitro modification with benzo(a)pyrene-diol-epoxide and transfer to LTK- cells as a eukaryotic test for carcinogens. 643 73

In order to facilitate cloning of genes for cell surface molecules, we cotransfected LTK- mouse fibroblasts with thymidine kinase (TK) genes and total human or mouse DNA. TK+ cells, selected by growth in HAT medium, were stained with fluorochrome conjugated monoclonal antibodies or other fluorescent ligands which bind to one or another membrane differentiation antigen or receptor. We isolated fluorescent transfectants expressing these molecules using a fluorescence activated cell sorter (FACS). For some antigens, spontaneous gene amplification occurred. By repeated cycles of FACS sorting and regrowth we obtained high expressing clones. We then isolated cDNA and genomic clones using selected cDNA probes to screen phage with cDNA inserts. DNA from virtually any tissue source transfected equally well for the various molecules except for DNA from a trophoblast derived choriocarcinoma cell line which did not transfect for Leu-2.
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PMID:Transfection and cloning of genes for membrane antigens using the FACS. 644 77

Thymidine kinase-deficient mouse L cells have been transformed with plasmid DNAs carrying 8-base-pair Xho I linker insertion mutations in the coding region of the herpes simplex virus type 1 thymidine kinase gene. When the mutant plasmids are introduced individually into LTK- cells, transformation efficiencies are greatly reduced relative to the wild type. However, when two mutant plasmids are cotransferred into the same LTK- recipients, significantly higher frequencies of transformation are observed (30-300 times). Here we demonstrate the usefulness of linker insertions for the study of homologous recombination in detecting the existence of normal thymidine kinase gene sequences (i.e., sequences lacking the insertions after recombination are substantiated by DNA . DNA hybridization). In addition, the frequencies of recombination in the various "crosses" are consistent with the known positions of the mutations.
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PMID:Novel use of synthetic oligonucleotide insertion mutants for the study of homologous recombination in mammalian cells. 657 60

A functional thymidine kinase (TK; ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) gene has been molecularly cloned from human DNA. The gene was rescued from a genomic library of TK-deficient mouse L cells transformed to the TK+ phenotype with total HeLa cell DNA. Of 14 overlapping clones, only one contained the intact human TK gene. The cloned recombinant bacteriophage carries a 16-kilobase insert derived entirely from human DNA and is capable of transforming LTK- cells to TK+ with an efficiency of 10 TK+ colonies per ng of DNA per 10(6) cells. Restriction endonuclease mapping shows that the functional human TK gene is at least twice as long as that reported for chicken. A 1.6-kilobase Xho I/EcoRI fragment was subcloned and found to hybridize to a human mRNA of 1.5 kilobases. When introduced into LTK- cells, the cloned human TK gene is regulated in the cell cycle-specific manner characteristic of TK+ mammalian cells. That is, TK activity in synchronized cells increases markedly with the onset of DNA synthesis. The signals governing the S-phase induction of TK activity reside within 16 kilobases of human DNA and are correctly interpreted by mouse cells.
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PMID:Molecular cloning and cell cycle-specific regulation of a functional human thymidine kinase gene. 657 46

In this report we present an experimental scheme that facilitates the study of homologous recombination between closely linked genes in cultured mammalian cells. Two different Xho I linker insertion mutants of the herpes simplex virus type 1 thymidine kinase (HTK) gene were introduced into mouse LTK- cells as direct repeats on a plasmid carrying a dominant selectable marker. Following stabilization of these sequences in the recipient cell, selection for TK+ was applied to detect recombinational events between different TK- genes. TK+ segregants were observed at a frequency of 10(-4)-10(-5) in lines harboring both mutant genes. Control lines carrying only one type of mutant HTK gene yielded TK+ cells at frequencies of 10(-7) or less. Physical analysis of the TK+ segregants has revealed the presence of an apparently normal HTK gene that is resistant to Xho l endonuclease digestion in each TK+ line examined. Analyses of the TK gene pairs before and after recombination suggest that at least 50% of the recombinants are the result of nonreciprocal exchanges of genetic information, or gene conversion events.
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PMID:Evidence for intrachromosomal gene conversion in cultured mouse cells. 657 76


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